Project Summary: The emerging preclinical and clinical evidence suggests that symptoms associated with alcohol withdrawal/negative affect is a primary risk factor for relapse and maintenance of alcohol use disorder (AUD). Anatomical structures comprising the extended amygdala, particularly the central nucleus of amygdala (CeA), are strongly implicated in anxiety and alcohol-drinking behaviors. Epigenetic mechanisms such as histone and DNA chemical modifications, have been shown to play important roles in the regulation of gene expression. This research component of CARE will examine how epigenetic modifications induced by chronic alcohol exposure and withdrawal at genome-wide level lead to an aberrant gene network pathway regulating various biological processes in the amygdala, producing anxiety-like behavior and escalated alcohol drinking. We propose the following Specific Aims: 1) To examine a) status of chromatin accessibility and loci of genomic epigenetic marks using ATAC-seq and H3K27me3 ChIP-seq in the amygdala of rats (male & female) during ethanol withdrawal after chronic ethanol exposure. The emerging data set will be merged with existing RNA-seq and ChIP-seq (H3K9/14ac mark) to identify integrated epigenetic regulation of differential gene expression in the amygdala. b) Dual specificity phosphatase 6 (Dusp6, novel gene identified by merger of H3K9/14ac ChIP-seq/RNA-seq) siRNA will attenuate anxiety-like behaviors via activating CBP and increasing histone acetylation of genes in the amygdala during ethanol withdrawal. 2) To examine if a) G9a siRNA infusion in the CeA will attenuate anxiety-like behaviors, normalize epigenetic and gene expression changes as well as deficits in synapses and dendritic spines in the amygdala of rats during ethanol withdrawal; b) Neuronal stimulation in the CeA using a chemogenetic (Gq coupled DREADD) approach will attenuate anxiety-like behaviors via restoring the proper balance in HDAC2/CBP regulation thereby leading to increased histone acetylation of target genes in the amygdala during ethanol withdrawal and c) Neuron-specific p300HAT guided by CRISPR-dCas9 infusion into CeA will increase histone acetylation at target genes, leading to increased gene expression in the amygdala, attenuation of anxiety-like behaviors and escalated drinking during withdrawal. 3) To examine whether CeA infusion of a HDAC2 and G9a siRNA will attenuate ethanol dependence-induced escalation in drinking in male and female rats, as measured by operant ethanol-self administration. 4) To translate epigenetic dynamics and expression of genes in the amygdala of alcohol dependent rats to postmortem amygdala of human alcoholics and correlate them to drinking data. Completion of proposed studies will provide new information on the epigenetic regulation of the whole transcriptome in the amygdala, leading to identification of molecular targets for the development of pharmacotherapy of AUD.